The spontaneous transfer of phospholipid molecules between membranes occurs by soluble monomer convection through the aqueous phase. The equilibrium partition between phospholipid monomers in the aqueous phase and in the membrane phase is correlated with the rate of phospholipid transfer. For this reason, the low aqueous solubility of phospholipids has been assumed to determine the rate of transfer of phospholipids between membranes. In this proposal evidence is presented that spontaneous intermembrane phospholipid transfer is limited by a large free energy of activation for monomer-membrane dissociation and association that is correlated with, but not determined by, the partition equilibrium. The goal of this project is to determine the value of this activation energy in different phospholipid monomer-membrane systems and its role in regulating phospholipid transfer between membranes. A technique based on resonance energy transfer (RET) will be used to monitor the rate of transfer of fluorescently labeled phospholipids between phospholipid vesicles. Measurements of the temperature dependence of the dissociation rate constant for the fluorescent phospholipids by the RET technique and measurements of the monomer-vesicle partition equilibrium by a centrifugation technique will be combined to construct a relative energy diagram for a monomer in vesicles, water, and transition states, that includes the relative contributions of enthalpy and entropy. The dependence of the transfer rate constants on pressure and vesicle size will be measured and used to formulate a mechanistic description of the dissociation-association barrier. If there is a large free energy barrier for phospholipid monomer-membrane dissociation and association, then phospholipid transfer proteins may act to facilitate the rate of monomer convection by reducing the height of this barrier. Consequently, the relative importance of this mechanism as compared to a shuttle-carrier mechanism will be determined for the activity of two bovine liver lipid transfer proteins (phosphatidylcholine-specific transfer protein and nonspecific phospholipid transfer protein). Measuring the rate of protein-dependent transfer across a dialysis membrane as well as kinetic modeling will be used for this purpose.